Article Cite This: Langmuir XXXX, XXX, XXX−XXX
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Easily Accessible Protein Nanostructures via Enzyme Mediated Addressing Arne A. Rüdiger,† Katharina Brassat,‡,§ Jörg K. N. Lindner,‡,§ Wolfgang Bremser,∥ and Oliver I. Strube*,† †
Paderborn University, Department of ChemistryBiobased and Bioinspired Materials, Warburger Str. 100, D-33098 Paderborn, Germany ‡ Paderborn University, Department of PhysicsNanostructuring, Nanoanalysis, and Photonic Materials, Warburger Str. 100, D-33098 Paderborn, Germany § Center for Optoelectronics and Photonics Paderborn (CeOPP), Warburger Str. 100, D-33098 Paderborn, Germany ∥ Paderborn University, Department of ChemistryCoatings, Materials and Polymers, Warburger Str. 100, D-33098 Paderborn, Germany ABSTRACT: Site-specific formation of nanoscaled protein structures is a challenging task. Most known structuring methods are either complex and hardly upscalable or do not apply to biological matter at all. The presented combination of enzyme mediated autodeposition and nanosphere lithography provides an easy-to-apply approach for the buildup of protein nanostructures over a large scale. The key factor is the tethering of enzyme to the support in designated areas. Those areas are provided via prepatterning of enzymatically active antidots with variable diameters. Enzymatically triggered protein addressing occurs exclusively at the intended areas and continues until the entire active area is coated. After this, the reaction self-terminates. The major advantage of the presented method lies in its easy applicability and upscalability. Largearea structuring of entire support surfaces with features on the nanometer scale is performed efficiently and without the necessity of harsh conditions. These are valuable premises for large-scale applications with potentials in biosensor technology, nanoelectronics, and life sciences.
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INTRODUCTION Nanostructures from Biological Polymers. Structured surfaces and coatings based on biological polymers are highly promising materials for manifold applications with regard to biocompatibility, biodegradability, sustainability, nontoxicity, and biofunctionality.1−5 While films and microstructures are easily accessible by manifold methods, structuring of surfaces with biomolecules on the sub-micrometer and nanometer scale is difficult to achieve. Frequently used methods for inorganic materials or synthetic polymers, such as lithography or etching techniques, mostly fail. This is because biopolymers are much more sensitive toward the commonly applied conditions in these processes. Especially proteins are sensitive to denaturation via thermal or mechanical stress, or exposure to irradiation and organic solvents. Though high precision structuring with such materials, even on the lower nanometer scale, is achievable by scanning probe techniques,6−8 its real-world application is hindered by severe drawbacks. The applied techniques are highly time, money, and energy consuming. Another approach is the combination of photolithography and biological self-assembly to obtain welldefined biological structures with complex geometries on the nanometer scale.9−11 However, the respective techniques are mostly applicable to only a limited variety of biomolecules, and © XXXX American Chemical Society
deposition of these structures onto surfaces results in arbitrary patterns. Moreover, all of these current approaches inherit essential issues, if structuring of larger surfaces or higher amounts, essential for any industrial application, is considered. An interesting alternative is the use of enzymes as a mediating agent to obtain structured surfaces with biomolecules and a high level of control, as most enzymes exhibit diameters of only a few nanometers and operate on the angstrom level. Their potential for nanostructuring has already been proven in combination with scanning probe techniques.12 However, again, these techniques are highly complex and hardly upscalable. In principle, though, enzymes are very promising candidates for the intended purpose of biomolecule nanostructuring. In addition to their specific catalyzing properties, they exhibit further beneficial attributes toward an environmentally friendly process. They are nontoxic, sustainable, and work under mild and energy efficient conditions. In summary, a structuring method that takes advantage of the specific enzyme properties, while being independent of Received: November 29, 2017 Revised: March 2, 2018
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DOI: 10.1021/acs.langmuir.7b04089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. Approach for site-specific deposition of single protein particles via enzyme mediated autodeposition with use of a prepatterned support, obtained by nanosphere lithography. Both steps are performed as hassle-free dip-coating processes. (GLYMO) was purchased from ABCR (Germany) and used without further purification. All other chemicals were received from local suppliers and used without further purification. Patterning of Supports via Nanosphere Lithography. Prior to the patterning of the support surface by nanosphere lithography, silicon (100) wafers with native SiO2 were cleaned in an oxygen/argon plasma (Oxford Instruments PlasmaLab 80 plus) with 2 sccm O2 and 8 sccm Ar at 75 mTorr with 50 W RF power for 5 min, resulting in a hydrophilization of the surface. A monolayer of polystyrene (PS) spheres was then deposited from aqueous suspension onto the Si/SiO2 surfaces. This is achieved by the doctor blade technique for which a droplet of the nanosphere suspension is drawn across the hydrophilic surface by a hydrophobic blade. In particular, a 40 μL droplet of the suspension was deposited on the surface, which was annealed to 26 °C, in an air atmosphere of 50% relative humidity. By this, hexagonally close-packed PS nanosphere masks were formed. The sphere masks were then modified by an oxygen/argon plasma under the same conditions as those used for the surface cleaning. The plasma treatment leads to a shrinking of the sphere diameter as PS is partially removed. The amount of removed material determines the final diameter of the shrunk spheres and can be adjusted by a variation of the plasma exposure time. In this work, larger PS spheres with an initial diameter of 618 nm were deposited on the support surface and shrunk in the plasma for 5 min to a diameter of approximately 380 nm, while 220 nm PS spheres were shrunk for 1.5 min to 160 nm. The modified monolayers of shrunk spheres were then used as shadow masks in nanosphere lithography. To this end, platinum was sputter deposited onto the sample, i.e., onto the shrunk spheres and the bare support surface between the shrunk spheres. Sputter deposition was performed in an “ISI PS-2” coating unit for 60 s at 1.2 kV and 20 μA under an argon atmosphere at 0.1 Torr. The PS spheres were subsequently dissolved by THF in an ultrasonic bath, resulting in a Pt thin film with free Si/SiO2 substrate areas at prior sphere positions, which are accessible for enzyme immobilization. Covalent Immobilization of Chymosin via GLYMO. Functionalization of the accessible native oxide layer of silicon wafer was achieved with GLYMO. To this, the support was immersed into a mixture of EtOH/H2O 80:20 (w/w) with a siloxane concentration of 10% (w/w) and 1% (w/w) triethylamine. The mixture was stirred at low rounds for 2 h at 20 °C. After this, it was allowed to stand unstirred for another 10 min. Functionalized supports were than rinsed multiple times with EtOH and DI water to remove nonspecifically adsorbed GLYMO. The supports were subsequently cured for 1 h at 110 °C, rinsed with additional EtOH and DI water, and dried. The following immobilization of chymosin was performed with 100 mL of a 1 M phosphate buffer at pH 7.0. Six mg of the freshly made enzyme formulation (62% solid enzyme content) was added to the buffer. This immobilization process was performed at 20 °C with gentle stirring. Enzyme coupling was monitored via activity tests as described in previous publications.13,17 Immobilization reaction was stopped after 96 h, and enzyme functionalized supports were washed with copious amounts of DI water to remove noncovalently bound chymosin and dried. Deposition of Casein with Immobilized Chymosin. The supports with covalently immobilized chymosin on the specific
complex methodology, is highly desirable to enable controlled and upscalable nanostructures of biopolymers. Nanostructuring via Enzyme Mediated Autodeposition. In this study, we introduce an easy-to-apply method for site-specific deposition of single biological particles, enabling structuring of surfaces on the nanometer scale. The approach is based on our original concept of enzyme mediated autodeposition (EMA).13−17 This technique combines industrially relevant and easy-to-apply autodeposition methods with the selectivity and specificity of enzymes as a trigger for particle deposition. The high level of deposition control originates from immobilization of the enzyme onto the desired support. The enzyme-functionalized support is then immersed into a precursor solution. Respective enzymatic reactions thus occur only in direct proximity to the support surface, which induces controlled deposition of the particles. The high practicability on the other hand derives from the execution procedure. The entire support is simply immersed two times, first into the enzyme solution and then into the protein dispersion. As a model system to obtain protein structures on the nanometer scale, the system casein/chymosin is utilized. Caseins are a group of proteins that make up the major protein content of milk. They have a long materials tradition as well as high potential for future applications.21−25 In an aqueous environment, the different caseins self-organize and form a complex aggregation structure, the casein micelle.26−29 The aspartic protease chymosin is able to specifically cleave the stabilizing hydrophilic parts of the protein micelle, which results in drastically changed solubility.30 Application of this cleavage reaction in EMA leads to destabilized micelles in direct proximity to the support, resulting in immediate deposition of the individual protein. The process is tunable to achieve a wide variety of deposition patterns, depending on the nature of enzyme immobilization.18−20 Though it was possible to get nanoscaled layer thicknesses, the lateral dimensions have been on the (sub)micrometer scale. To obtain genuine, three-dimensional nanostructures, a preceding step to prepare defined enzyme patterns is required. To sustain easy applicability and convenient execution of the overall process, prepatterning of the support with functional groups, applicable as enzyme binding sites, is the method of choice. Such a support can simply be immersed into the enzyme solution for immobilization, and afterward into the protein dispersion for particle deposition (Figure 1).
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EXPERIMENTAL SECTION
Materials. Polystyrene spheres (CV < 2%, 2% (w/v)) were obtained from Microparticles Inc. and were used as received. Casein from bovine milk, chymosin, and hemoglobin were purchased from Sigma-Aldrich (Germany). (3-Glycidoxypropyl)trimethoxysilane B
DOI: 10.1021/acs.langmuir.7b04089 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 2. SEM images and respective model sketches for each step of nanosphere lithography. SiO2 areas are shown in blue color. (a) Self-assembled hexagonally close-packed PS sphere monolayer; (b) shrunk spheres after plasma treatment; (c) sputtering with Pt; (d) subsequent removal of PS spheres yields antidot structured Pt film with free SiO2 dots.
Figure 3. (a) SEM images of s representative section (100 μm2) and magnification showing uniformity of the antidot distribution over the entire support with defects
